Gram-positive bacteria belonging to the genus Rhodococcus, some of which were formerly classified as Nocardia, Mycobacterium, Gordona, or Jensenia spp., or as members of the “rhodochrous” complex, are widely distributed in the environment. Members of the genus Rhodococcus exhibit a wide range of metabolic activities, including antibiotic and amino acid production, biosurfactant production, and biodegradation and biotransformation of a large variety of organic and xenobiotic compounds (see Vogt Singer and Finnerty, 1988, J. Bacteriol., 170:638-645; Quan and Dabbs, 1993, Plasmid, 29: 74-79; Warhurst and Fewson, 1994, Crit. Rev. Biotechnol., 14:29-73). Unfortunately, few appropriate genetic tools exist to investigate and exploit these metabolic activities in Rhodococcus and like organisms (see Finnerty, 1992, Annu. Rev. Microbiol., 46:193-218).
Recently, several Rhodococcus plasmids and Rhodococcus-Escherichia coli shuttle vectors have been described. These plasmids and vectors can be divided into five different derivation groups: a) plasmids derived from Rhodococcus fascians (Desomer et al., 1988, J. Bacteriol., 170:2401-2405; and Desomer et al., 1990, Appl. Environ. Microbiol., 56:2818-2815); b) plasmids derived from Rhodococcus erythropolis (JP 10248578; EP 757101; JP 09028379; U.S. Pat. No. 5,705,386; Dabbs et al., 1990, Plasmid, 23:242-247; Quan and Dabbs, 1993, Plasmid, 29:74-79; Dabbs et al., 1995, Biotekhnologiya, 7-8:129-135; De Mot, et al., 1997, Microbiol., 143:3137-3147); c) plasmids derived from Rhodococcus rhodochrous (EP 482426; U.S. Pat. No. 5,246,857; JP 1990-270377; JP 07255484; JP 08038184; U.S. Pat. No. 5,776,771; EP 704530; JP 08056669; Hashimoto et al., 1992, J. Gen. Microbiol., 138:1003-1010; Bigey et al., 1995, Gene, 154:77-79; Kulakov et al., 1997, Plasmid, 38:61-69); d) plasmids derived from Rhodococcus equi (U.S. Pat. No. 4,920,054; Zheng et al., 1997, Plasmid, 38:180-187) and e) plasmids derived from a Rhodococcus sp. (WO 89/07151; U.S. Pat. No. 4,952,500; Vogt Singer et al., 1988, J. Bacteriol., 170:638-645; Shao et al., 1995, Lett. Appl. Microbiol., 21:261-266; Duran, 1998, J. Basic Microbiol., 38:101-106; Denis-Larose et al., 1998, Appl. Environ. Microbiol., 64:4363-4367).
While these prior studies describe several plasmids and shuttle vectors, the relative number of commercially available tools that exist for the genetic manipulation of Rhodococcus and like organisms remains limited. One of the difficulties in developing a suitable expression vector for Rhodococcus is the limited number of sequences encoding replicase or replication proteins (rep) which allow for plasmid replication in this host. Knowledge of such sequences is needed to design a useful expression or shuttle vector. Although replication sequences are known for other shuttle vectors that function in Rhodococcus (see for example Denis-Larose et al., 1998, Appl. Environ. Microbiol., 64:4363-4367); Billington, et al., J. Bacteriol. 180 (12), 3233-3236 (1998); Dasen, G. H. GI:3212128; and Mendes, et al, GI:6523480) they are rare.
Similarly, another concern in the design of shuttle expression and shuttle vectors in Rhodococcus is plasmid stability. The stability of any plasmid is often variably and maintaining plasmid stability in a particular host usually requires the antibiotic selection, which is neither an economical nor a safe practice in the industrial scale production. Little is known about genes or proteins that function to increase or maintain plasmid stability without antibiotic selection.
The problem to be solved, therefore is to provide additional useful plasmid and shuttle vectors for use in genetically engineering Rhodococcus and like organisms. Such a vector will need to have a robust replication protein and must be able to be stably maintained in the host.
Applicants have solved the stated problem by isolating and characterizing a novel cryptic plasmid, pAN12, from Rhodococcus erythropolis strain AN12 and constructing a novel Escherichia coli-Rhodococcus shuttle vector using pAN12. Applicants' invention provides important tools for use in genetically engineering Rhodococcus species (sp.) and like organisms. The instant vectors contain a replication sequence that is required for replication of the plasmid and may be used to isolate or design other suitable replication sequences for plasmid replication. Additionally, the instant plasmids contain a sequence having homology to a cell division protein which is required for plasmid stability. Applicants' shuttle vectors are particularly desirable because they are able to coexist with other shuttle vectors in the same Rhodococcus host cell. Therefore, Applicants' vectors may also be used in combination with other compatible plasmids for co-expression in a single host cell.